Ingeborg Hanbauer

Sodium‐Sensitive Cocaine Binding to Rat Striatal Membrane: Possible Relationship to Dopamine Uptake Sites

Abstract: In rat striatal membranes, NaCl induced a twofold increase in the maximal number of cocaine binding sites but did not alter the affinity of these sites for cocaine. This effect was concentration‐dependent, specific to sodium ions, and occurred in membranes prepared from corpus striatum but not from other brain regions. Lesions with 6‐hydroxydopamine but not with kainic acid eliminated the sodium‐induced increase in binding and produced a decrease in the Bmax of binding measured in the presence of NaCl. The capacity of a series of drugs to interfere with Na+–dependent cocaine binding correlated well with their capacity to inhibit [3H]dopamine uptake into rat striatal synaptosomes. The present results suggest that Na+–dependent cocaine binding sites are localized presynaptically on dopaminergic nerve terminals in corpus striatum, and may be related to dopamine uptake sites.

Role of nitric oxide in NMDA-evoked release of [3H]-dopamine from striatal slices

Evidence that excitatory amino acids act via N-methyl-D-aspartate (NMDA) receptors to evoke the release of catecholamines from axonal terminals and synaptosomes has been used to argue for the presence of pre-synaptic NMDA receptors. NMDA receptor agonists also generate nitric oxide (NO) which rapidly diffuses through neural tissue. We find that exogenously applied NO evokes [3H]-dopamine release from cultured neurons. This release is not blocked by the NMDA antagonist MK-801 nor by tetrodotoxin. Both NG-nitroarginine which inhibits NO synthesis, and hemoglobin which binds extracellular NO, block NMDA-evoked [3H]-dopamine release from striatal slices. A major role of endogenously-synthesized NO may be to evoke neurotransmitter release in local volumes of neural tissue.

Dopamine Uptake is Differentially Regulated in Rat Striatum and Nucleus Accumbens

Abstract: Active uptake of 3,4‐dihydroxyphenylethylamine (dopamine) is sodium‐ and temperature‐dependent, strongly inhibited by benztropine and nomifensine, and present in corpus striatum and nucleus accumbens. In rat striatum dopamine uptake is related to a receptor that is specifically labelled by [3H]cocaine in the presence of Na+ and is located on dopaminergic terminals. The dopamine uptake is differentially affected in the two areas by single or repeated injections of cocaine. Cocaine inhibits dopamine uptake in slices of corpus striatum. Moreover Na+‐dependent [3H]cocaine binding is not detectable in nucleus accumbens. Nomifensine inhibits [3H]dopamine uptake by interacting with low‐ and high‐affinity sites in corpus striatum, but shows only low affinity for dopamine uptake in nucleus accumbens. The present data indicate that different mechanisms are involved in the regulation of dopamine uptake in corpus striatum and nucleus accumbens.

Nitric Oxide Protects against the Cytotoxic Effects of Reactive Oxygen Species

Reactive oxygen species (ROS) have been thought to be involved in the etiology of various degenerative diseases.1,2 ROS are derived from the reaction between hydrogen peroxide and metals generating powerful oxidants such as metallo-oxo species or hydroxyl r a d i ~ a l . ~ . ~ These reactive species have been shown to oxidize DNA, proteins, and lipids and thereby can ultimately cause cell death.2 Among free-radical molecules, nitric oxide (NO) has been shown to participate in the regulation of various physiological functions.’ Prior to this discovery, NO was thought to be a toxic chemical associated with other reactive nitrogen oxide species in air pollution and cigarette smoke.6 Endogenous NO is formed from a class of enzymes known as nitric oxide synthases (NOS). The constituent form of this enzyme generates low levels of NO which activate enzymes such as guanylate cyc la~e .~ The other isozyme of NOS, referred to as the inducible form, is capable of generating large amounts of NO for prolonged periods of time.’ Some studies suggest that tissue injury during ischemia reperfusion is attributed to endogenously generated However, under these conditions, reactive oxygen species derived from hydrogen peroxide or superoxide are also present.’-” While some studies have attributed neuronal death mediated by excitatory amino acids to NO, other studies suggest NO is protective.I2Jj This report extends the studies recently published from our laboratoryIz on the role NO might play in cytotoxicity in the presence of reactive oxygen intermediates.

Effects of hypoxia lasting up to one month on the catecholamine content in rat carotid body.

Long-term exposure to hypoxia (10%O2 + 90%N2) elicits an increase in the dopamine content of the carotid body after 2 days and in the noradrenaline content after one week. When hypoxia is extended to 4 weeks the dopamine and noradrenaline content increase 15- and 12-fold, respectively. These changes are associated with an increase in size and protein content of the carotid body. Removal of the superior cervical ganglion, which causes a 50% reduction of the noradrenaline content, fails to prevent the increase in amine content elicited by hypoxia. Exposure to hypoxia for 4 weeks slightly increases the dopamine and 3,4-dihydroxyphenylacetic acid content in superior cervical ganglia and that of dopamine in adrenal glands, but fails to alter the levels of noradrenaline, adrenaline and 3-methoxy-4-hydroxyphenylglycol in heart and adrenal glands, that of noradrenaline in superior cervical ganglia and that of dopamine and 3,4-dihydroxyphenylacetic acid in the caudate nucleus. From these data it can be inferred that the increase in dopamine and noradrenaline content occurs as a specific response to long-term hypoxia and may involve both induction of tyrosine hydroxylase and hyperplasia of glomus cells of carotid bodies.

Secretion of enkephalin-like peptides from canine adrenal gland following splanchnic nerve stimulation

Plasma obtained from dogs implanted with an indwelling catheter in the left adrenal lumbar vein contains a greater amount of (met5)-enkephalin-like peptides than does plasma sampled from jugular and femoral veins or femoral artery. Stimulation of the splanchnic nerve ipsilateral to the cannulated vein results in a voltage dependent rise in blood pressure and an increase in the content of (met5)-enkephalin-like peptides in the lumbar vein plasma. Gel filtration reveals that this increase can be accounted by a greater amount of the low molecular weight enkephalin-like peptide(s). Analysis of the material present in this chromatographic peak by high voltage paper electrophoresis followed by chromatography reveals two peptides, one of them having a migration distance and Rf similar to that for the (met5)-enkephalin standard. This study shows the (met5)-enkephalin-like peptides can be released into the circulation following splanchnic nerve stimulation of the adrenal gland.

Selective decrease of dopamine content in rat carotid body during exposure to hypoxic conditions.

The rat carotid body contains norepinephrine (NE), dopamine (DA) and serotonin (5-HT)4,5, 8. Each of these amines is stored in membrane-bound granules located in the cystoplasm of type I cells which are the most abundant cells in the rat carotid body 1. Although the role of catechotamines in chemoreceptor function remains to be elucidated, some reports have shown that in the cat, NE and DA inhibit chemoreceptor dischargOl,lL In rat carotid body, exposure to hypoxia causes a delayed increase in tyrosine hydroxylase activity without a concomitant change in dopamine-beta-hydroxytase 3. This finding suggests that during hypoxia, DA rather than NE, functions in chemoreceptor regulation. To elucidate which catecholamine participates in chemoreceptor function during hypoxia, we measured DA and NE content of the carotid body at various times after hypoxia. We used rats with unilateral transection of the sinus nerve, which was pertormed 20 days before the experiment. A desiccator (30 cm in diameter) was connected to a gas tank containing either air or a gas mixture of 5 ~ 02 and 95 ~ N2. The gas mixture was allowed to flow for 30 min through a desiccator containing 3 male Sprague-Dawley rats, weighing about 170 g. The rats were killed by cervical dislocation either immediately after the exposure to hypoxia or after they were placed for various times in room air following the hypoxia. Both carotid bodies were rapidly dissected and the catecholamines assayed by mass fragmentography as previously reported 2,5. Deuterium labeled DA ~.DA-dT) and alpha-methylnorepinephrine ~a-MNE) were used as internal standards for DA and NE respectively. The catecholamines were acylated with pentafluoropropionic anyhdride 5 and the following m/e ratios were focused: a-MNE, 190; NE, 176; DA, 176; DA-dT, 178. The catecholamine derivatives were chromatographed on a 9 ft. glass column packed with 3 ~o OV17 on Gas Chrom Q with the flash heater at 250 °C, the oven at 150 °C and a helium flow of 35 ml/min. The retention times of the various amines expressed in seconds were: a-MNE, 120; NE, 160; DA-d7 and DA, 320.

Accumulation of adenosine 3′,5′-monophosphate induced by catecholamines in the rat superior cervical ganglionin vitro

Abstract Levels of cyclic AMP are elevated rapidly in rat superior cervical ganglia incubated in medium containing low concentrations of isoprenaline, adrenaline and noradrenaline. Phenylephrine and dopamine increase cyclic AMP when used at higher concentrations. The moderate rise induced by dopamine has a slow onset and long duration; the peak accumulation induced by beta-adrenergic agents is rapid and short-lasting. Preincubation with propranolol completely suppresses the elevation of cyclic AMP levels induced by all the agents tested.

THE ROLE OF NERVE GROWTH FACTOR IN THE RAMIFICATION OF SYMPATHETIC NERVE FIBRES INTO THE RAT IRIS IN ORGAN CULTURE

Small amounts of nerve growth factor (NGF) were present in the superior cervical ganglion and the iris of the rat. The observations that NGF content in each of the tissues was depleted during organ culture and that more NGF appeared in the media than was originally present in the tissues indicated that synthesis or activation of NGF had occurred in organ culture. Antibody to NGF or the depletion of endogenous NGF retarded growth of new sympathetic axons into irides in organ culture. Exogenously added NGF appeared to enhance the initiation of axonal sprouting and the rate of the ramification of nerve fibres.

ROLE OF CALMODULIN IN DOPAMINERGIC TRANSMISSION

Abundant evidence indicates that in brain a great number of dopamine receptors are coupled with adenylate cyclase. Therefore, one of the most prominent physiological responses elicited by dopamine is an increased synthesis of cAMP due to the stimulation of a dopamine receptor-coupled adenylate cyclase [for review see Reference 1). Hence, adenylate cyclase can be viewed as a subunit of the supramolecular structure of the dopamine receptor. In general, the recognition site for a neurotransmitter with adenylate cyclase is coupled by guanine nuc leo t ide~~ .~ via a specific GTP-binding protein.’ In the case of striatal dopamine receptors it was speculated that this protein probably also modulates the affinity of the recognition site to d ~ p a m i n e . ~ However, the GTP-binding protein is not the only membrane protein that modulates dopamine receptor function. Increasing evidence indicates that calmodulin, a thermostable Ca2+ binding protein, also functions as a modulator of the Ca”-dependent adenylate cyclase.6 protein k i n a ~ e . ~ and phosphodiestera~e~,~ and. therefore, at least in nervous tissue participates in receptor function. In brain two forms of calmodulin appear to be present: a soluble form and membrane-hound form. Studies on subcellular distribution of calmodulin show that this protein is preferentially located in the synaptosome-rich pellet with lesser amounts in the supernatant fraction [~ytosol).’~ With regard to the relationship of calmodulin to dopamine function, it is important to note that the membrane-bound form of calmodulin appears to function with adenylate cyclase, whereas the soluble form is required for phosphodiesterase activation. While there is a more immediate relationship between dopamine. adenylate cyclase, and calmodulin. the relationship between dopamine, calmodulin, and phosphodiesterase is remote. Substantial evidence indicates that calmodulin is involved in the dopamine receptor function by regulating the coupling of the membrane-bound adenylate cyclase to the transmitter recognition site.” In response to persistent dopamine receptor stimulation i t appears to also regulate the link between increased cAMP content and phosphodiesterase a~tivation.”~’~ Hence, calmodulin can be viewed as an important component in the transduction of short-term and long-term stimulation of specific post-synaptic dopamine receptors.